CHD3 gene detection method and application thereof

Abstract

The invention provides a CHD3 gene detection method and application thereof, and belongs to the technical field of gene detection.The method comprises the following steps that a blood or ovarian tissue sample of a patient is collected, DNA extraction and PCR amplification are conducted on the blood or tissue sample, a CHD3 specific primer is designed, a target fragment is amplified, the expression level of the CHD3 gene is detected, and data analysis is conducted. And comparing and analyzing the detection result with a normal control group. By detecting the expression level of the CHD3 gene in the blood or tissue sample of a patient, the early occurrence of the ovarian cancer can be effectively judged. The method has the characteristics of high sensitivity, strong specificity, simplicity and convenience in operation and the like, can obviously improve the early diagnosis rate of ovarian cancer, and gains precious time for treatment of patients. The invention provides an innovative solution for early screening and diagnosis of ovarian cancer, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, and in particular to a CHD3 gene detection method and application thereof. BACKGROUND

[0002] Ovarian cancer is one of the common malignant tumors in women. Since its early symptoms are not obvious, most patients are in the advanced stage when diagnosed, resulting in poor treatment effect and poor prognosis.

[0003] The biological function of CHD3 gene is associated with diseases. CHD3 encodes Chromodomain Helicase DNA-Binding Protein 3, which is a core component of the NuRD complex. Its functions include: chromatin remodeling: regulating histone deacetylation (such as HDAC1 / 2) through ATP-dependent helicase activity, affecting gene transcription activation or inhibition. Cell cycle regulation: involved in centrosome anchoring and spindle assembly, maintaining normal mitosis. Developmental regulation: plays a key role in neural development, heart formation and left-right body axis establishment.

[0004] CHD3 mutation causes autosomal dominant disease, characterized by intellectual disability, macrocephaly, facial deformity (such as prominent forehead, wide eye spacing), low muscle tone and autism spectrum disorder. De novo mutations in chromatin remodeling genes (including CHD3) account for 10% of severe congenital heart disease, associated with left ventricular outflow tract obstruction and other lesions. However, there is not much research on the expression detection and inhibition of CHD3 gene. Therefore, a CHD3 gene detection method and application thereof need to be designed. It can significantly improve the early diagnosis rate of ovarian cancer, win valuable time for the treatment of patients, and has important clinical application value. SUMMARY

[0005] The purpose of the present application is to provide a CHD3 gene detection method and application thereof, which solves the technical problem that there is not much research on the inhibition of CHD3 gene on cancer cells.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A CHD3 gene detection method, the method comprising the following steps:

[0008] Step 1: collecting blood or ovarian tissue samples of patients;

[0009] Step 2: DNA extraction of blood or tissue samples;

[0010] Step 3: PCR amplification, design CHD3 specific primers, and amplify target fragments;

[0011] Step 4: Detect the expression level of CHD3 gene;

[0012] Step 5: Data analysis, compare the test results with the normal control group.

[0013] Further, 24 hours before blood collection, avoid alcohol and intense exercise, and keep calm for at least 5 minutes. The blood should be sent for examination within 2 hours at room temperature, and should not be frozen. Tissue samples should be frozen at -196℃ in liquid nitrogen or soaked in RNAlater. Paraffin sections should be deparaffinized in xylene and dehydrated in gradient ethanol after formaldehyde fixation.

[0014] Further, in step 2, if it is a blood sample, it should be anticoagulated. Use EDTA or heparin sodium anticoagulant tubes to collect whole blood, avoid blood clotting, otherwise the cell nucleus will be lost. Fresh samples should be stored at 4℃ for a short period of time. If the storage time exceeds the specified time, it needs to be frozen at -70℃ or in liquid nitrogen. Red blood cells should be lysed by adding lysis solution or Triton X-100 / SDS. Centrifuge to remove the supernatant and retain the white blood cell precipitate.

[0015] If it is a tissue sample, it should be first crushed. The liquid nitrogen grinding method is used to take 5-50 mg of tissue. After freezing in liquid nitrogen, the mortar is ground into powder. Then, the powder is quickly transferred to the lysis solution. RNase A with a concentration of 100 μg / mL is added for 5 hours of incubation at 37℃ to degrade RNA.

[0016] Add magnetic beads and binding liquid, mix well and adsorb DNA for 5 minutes. After magnetic separation, wash the magnetic beads with ethanol for 2-4 times. Add 100 μL of elution solution to the magnetic beads and incubate at room temperature for 2 minutes.

[0017] Further, the specific process of step 3 is as follows: CHD3 gene sequence acquisition and target region selection. Access the NCBI Gene database, input CHD3 and select the target species. In the Genomic regions tab of the gene page, copy the FASTA format sequence of the coding region or specific exon region. If specific functional domains need to be amplified, the start and end positions in CDS need to be marked. Check for repeat sequences, pseudogenes or homologous genes through the UCSC Genome Browser to avoid non-specific regions.

[0018] Use Primer3 Plus to design. Paste the CHD3 target sequence into Primer3 Plus and set the parameters. The target fragment length is 300-500 bp, and the salt concentration is 58-62℃. Click Pick Primers to select primer pairs with high Tm matching degree and no secondary structure. F: 5'-CAGCAACCACATCCTCAACG-3', R: 5'-TGGTGTTGCTGCTGAGTTCT-3'.

[0019] The reaction system is: pre-denaturation 95 DEG C, 5 min, denaturation 95 DEG C, 30 s, annealing Tm-5 DEG C, 30 s, extension 72 DEG C, 1 min / kb, final extension 72 DEG C, 5 min, and the cycle number is 30-35.

[0020] 5% agarose gel electrophoresis, the expected band size should meet the design, no mixed band or primer dimer, recover the target band and sequence, compare with the CHD3 reference sequence, and verify the amplification accuracy.

[0021] Further, the specific process of step 4 is: in step 5, a set of blank control groups are designed, and the experimental operation is the same as that of the sample; the blood or ovarian tissue sample of the blank control group is collected from a normal woman, and then the expression level of the CHD3 gene between the two groups is compared; if the number of the CHD3 gene is less, then the CHD3 gene artificially inhibits tumor cells.

[0022] The application of the CHD3 gene is used for inhibiting ovarian cancer tumors.

[0023] The application has the following beneficial effects due to the adoption of the above technical scheme:

[0024] The CHD3 gene expression level in the blood or tissue sample of the patient can be detected by the method, so that the early occurrence of ovarian cancer can be effectively judged. The method has the characteristics of high sensitivity, strong specificity and simple operation, and can significantly improve the early diagnosis rate of ovarian cancer, and win valuable time for the treatment of patients. The application provides an innovative solution for the early screening and diagnosis of ovarian cancer, and has important clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a method flowchart of the application. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the application more clear and obvious, the application will be further described in detail below with reference to the drawings and preferred embodiments.

[0027] As Figure 1 shown, a CHD3 gene detection method, the method comprises the following steps:

[0028] Step 1: Collect blood or ovarian tissue samples from patients. Avoid alcohol, strenuous exercise, and emotional stability for at least 24 hours before blood collection. Rest for at least 5 minutes before blood collection. Blood samples should be sent for examination within 2 hours at room temperature and should not be frozen. Tissue samples should be frozen in liquid nitrogen at -196°C or soaked in RNAlater. Paraffin sections should be deparaffinized with xylene and dehydrated with gradient ethanol after formaldehyde fixation.

[0029] Step 2: Extract DNA from blood or tissue samples. If it is a blood sample, use EDTA or heparin sodium anticoagulant tubes to collect whole blood and avoid clotting, which can cause cell nucleus loss. Fresh samples should be stored at 4°C for short-term storage. If the storage time exceeds the specified time, the sample should be stored at -70°C or in liquid nitrogen. Lysis of red blood cells can be achieved by adding lysis solution or Triton X-100 / SDS. Centrifuge to remove the supernatant and retain the white blood cell pellet.

[0030] If it is a tissue sample, first crush it. Liquid nitrogen grinding method: take 5-50 mg of tissue, grind it into powder after freezing in liquid nitrogen, quickly transfer it to lysis solution, and incubate it at 37°C for 5 hours with RNase A at a concentration of 100 μg / mL to degrade RNA.

[0031] Add magnetic beads and binding solution, mix well and adsorb DNA for 5 minutes. After magnetic separation, wash the magnetic beads with ethanol for 2-4 times. Add 100 μL elution solution to the magnetic beads and incubate at room temperature for 2 minutes.

[0032] Step 3: PCR amplification, design CHD3 specific primers and amplify target fragments. Obtain CHD3 gene sequence and select target region. Access NCBI Gene database, input CHD3 and select target species. In the Genomic regions tab of the gene page, copy the FASTA format sequence of the coding region or specific exon region. If you need to amplify a specific functional domain, you need to mark the start and end positions in the CDS. Check for repeat sequences, pseudogenes, or homologous genes through the UCSC Genome Browser to avoid non-specific regions.

[0033] Use Primer3 Plus to design and paste the CHD3 target sequence into Primer3 Plus. Set the target fragment length to 300-500 bp and the salt concentration to 58-62°C. Click Pick Primers to select primers with high Tm matching and no secondary structure. F: 5'-CAGCAACCACATCCTCAACG-3', R: 5'-TGGTGTTGCTGCTGAGTTCT-3'.

[0034] The reaction system is as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at Tm-5°C for 30 s, extension at 72°C for 1 min / kb, and final extension at 72°C for 5 min. The number of cycles is 30-35.

[0035] Perform 0.5% agarose gel electrophoresis. The expected band size should meet the design, with no extraneous bands or primer dimers. Recover the target band and sequence it. Compare it with the CHD3 reference sequence to verify the amplification accuracy.

[0036] Step 4: Detect the expression level of the CHD3 gene;

[0037] Step 5: Data analysis. Compare the test results with the normal control group. Design a blank control group. The experimental procedure is the same as that for the samples. Blood or ovarian tissue samples are collected from normal women in the blank control group. Then compare the expression levels of the CHD3 gene between the two groups. If the number of CHD3 genes is lower, it is assumed that the CHD3 gene inhibits tumor cells.

[0038] This invention discloses an application of the CHD3 gene to suppress ovarian cancer tumors. By applying the CHD3 gene, ovarian cancer tumors can be inhibited, and the early occurrence of ovarian cancer can be effectively determined by detecting the expression level of the CHD3 gene in the patient's blood or tissue samples. This method features high sensitivity, strong specificity, and simple operation, significantly improving the early diagnosis rate of ovarian cancer and saving valuable time for patient treatment. This invention provides an innovative solution for the early screening and diagnosis of ovarian cancer and has significant clinical application value.

[0039] Sample collection: Blood or ovarian tissue samples are collected from the patient. CHD3 gene expression detection: The expression level of the CHD3 gene is detected using real-time quantitative PCR technology. Data analysis: The test results are compared and analyzed with a normal control group. Diagnostic results output: Based on the analysis results, the early occurrence of ovarian cancer is determined.

[0040] Matters not covered in this invention are common knowledge.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the CHD3 gene, characterized in that: The method includes the following steps: Step 1: Collect blood or ovarian tissue samples from the patient; Step 2: Extract DNA from blood or tissue samples; Step 3: PCR amplification, designing CHD3-specific primers to amplify the target fragment; Step 4: Detect the expression level of the CHD3 gene; Step 5: Data analysis, compare and analyze the test results with the normal control group.

2. The method for detecting the CHD3 gene according to claim 1, characterized in that: In step 1: Refrain from alcohol and strenuous exercise for 24 hours before blood collection. After stabilizing your emotions, rest for ≥5 minutes. Send the blood sample to the laboratory at room temperature within 2 hours. Avoid freezing. Quick-freeze tissue samples in liquid nitrogen at -196℃ or soak them in RNAlater. Perform paraffin sectioning, formaldehyde fixation, xylene dewaxing, and graded ethanol dehydration.

3. The method for detecting the CHD3 gene according to claim 1, characterized in that: In step 2, if the sample is blood, anticoagulation treatment is required. Whole blood is collected using EDTA or heparin sodium anticoagulation tubes to avoid clotting, which would otherwise lead to cell nucleus loss. Fresh samples should be stored at 4°C for a short period. If the storage time exceeds the set time, the samples should be frozen at -70°C or in liquid nitrogen. Red blood cells are lysed by adding lysis buffer or Triton X-100 / SDS, centrifuged to remove the supernatant, and the white blood cell precipitate is retained. If it is a tissue sample, first crush it using liquid nitrogen grinding. Take 5–50 mg of tissue, freeze it in liquid nitrogen, grind it into powder in a mortar and pestle, quickly transfer it to lysis buffer, add 100 μg / mL RNase A, and incubate at 37°C for 5 hours to degrade RNA. Add magnetic beads and binding buffer, mix well and adsorb DNA for 5 minutes. After magnetic separation, wash 2–4 times with ethanol. Add 100 μL of elution buffer to the magnetic beads and incubate at room temperature for 2 minutes.

4. The method for detecting the CHD3 gene according to claim 1, characterized in that: Step 3 is as follows: CHD3 gene sequence acquisition and target region selection. Access the NCBI Gene database, enter CHD3 and select the target species. In the Genomic regions tab of the gene page, copy the FASTA format sequence of the coding region or specific exon region. If a specific functional domain needs to be amplified, the start and end positions in the CDS need to be marked. Check for repetitive sequences, pseudogenes or homologous genes through the UCSC Genome Browser to avoid non-specific regions. Using Primer3 Plus, the CHD3 target sequence was pasted into Primer3 Plus. The parameters were set as follows: target fragment length 300-500bp, salt concentration 58-62℃. Then, the "Pick Primers" button was clicked to select primer pairs with high Tm matching and no secondary structure: F: 5′-CAGCAACCACATCCTCAACG-3′, R: 5′-TGGTGTTGCTGCTGAGTTCT-3′. The reaction system was as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at Tm-5℃ for 30 s, extension at 72℃ for 1 min / kb, final extension at 72℃ for 5 min, with a cycle number of 30-35. Perform 5% agarose gel electrophoresis. The expected band size should meet the design, with no extraneous bands or primer dimers. Recover the target band and sequence it. Compare it with the CHD3 reference sequence to verify the amplification accuracy.

5. The method for detecting the CHD3 gene according to claim 1 and its application, characterized in that: The specific process of step 4 is as follows: In step 5, a blank control group is designed. The experimental operation is the same as that of the sample. Blood or ovarian tissue samples are collected from normal women in the blank control group. Then, the expression level of CHD3 gene between the two groups is compared. If the number of CHD3 genes is low, it is assumed that CHD3 gene inhibits tumor cells.

6. An application of the CHD3 gene, characterized in that: The CHD3 gene is used to suppress ovarian cancer tumors.

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